Monoclonal antibodies specific for the pb2 antigen of the human influenza virus (flu), nucleotide sequences, method and diagnostic kit for flu infection

ABSTRACT

Generation of monoclonal antibodies, or fragments thereof, that recognize the PB2 protein of the human influenza virus (Flu), where the monoclonal antibodies or fragments thereof, has a heavy chain variable region and light chain variable region. Furthermore, a diagnostic method is provided to detect Flu infections in biological samples of nasopharyngeal secretions, using monoclonal antibodies in diagnostic kit format.

DESCRIPTION OF THE INVENTION

Monoclonal antibodies, or fragments thereof are disclosed, thatrecognize the PB2 protein of the human influenza virus (Flu), where saidmonoclonal antibodies or fragments thereof comprise an antibody thatcomprises a light chain variable region where its CDR1 (CDR_(LC1)) isdefined according to SEQ ID NO: 1, its CDR2 (CDR_(LC2)) is defined bySEQ ID NO: 2 and its CDR3 (CDR_(LC3)) corresponds to SEQ ID NO: 3, and aheavy chain variable region where its CDR1 (CDR_(HC1)) is definedaccording to SEQ ID NO: 4, its CDR2 (CDR_(HC2)) is defined by SEQ ID NO:5 and its CDR3 (CDR_(HC3)) corresponds to SEQ ID NO: 6, or an antibodycomprising a light chain variable region where its CDR1 (CDR_(LC1)) isdefined according to SEQ ID NO: 7, its CDR2 (CDR_(LC2)) is defined bySEQ ID NO: 8 and its CDR3 (CDR_(LC3)) corresponds to SEQ ID NO: 9, and aheavy chain variable region where its CDR1 (CDR_(HC1)) is definedaccording to SEQ ID NO: 10, its CDR2 (CDR_(HC2)) corresponds to SEQ IDNO: 11 and its CDR3 (CDR_(HC3)) corresponds to SEQ ID NO: 12, where saidantibody can be used as detection or capture antibody. Additionally, amethod for diagnosing Flu infection in a biological sample is providedthat uses monoclonal antibodies in diagnostic kit format to detect Flu,where said kit comprises at least one monoclonal antibody against Flu aspreviously described.

FIELD OF THE INVENTION

The present invention relates to monoclonal antibodies, or fragmentsthereof, that recognize the PB2 protein of the human influenza virus,useful for the development of diagnostic methods of influenza infectionin humans.

BACKGROUND OF THE INVENTION

Influenza is an infectious disease of the respiratory tract caused bythe human influenza virus. This virus is responsible for producingsevere or mild respiratory symptoms, mainly affecting the areas of thenose, throat, bronchial tubes and occasionally the lungs. In general,clinical symptoms of influenza are similar to those of seasonal flu,however, symptoms can be variable and range from an asymptomaticinfection to severe pneumonia that can lead to death.¹. The virus iseasily transmitted from person to person or through drops or smallparticles that have been expelled through the cough or sneeze of a sickperson, which makes it spread quickly and be part of seasonal epidemics.¹1. http://www.who.int/csr/disease/swineflu/faq/es/#doesit

Influenza virus can be detected throughout the year, but its detectionincreases in the autumn-winter season, although time and duration can bevariable.². According to epidemiological statistics, in the UnitedStates in 2016, 310,000 people were hospitalized for complicationsrelated to influenza. In the same country, statistics indicate that thisinfection causes around 89,000 deaths annually. From the point of viewof economic cost, losses due to human influenza viruses in the UnitedStates are estimated to reach an annual cost that ranges from 71 to 150billion dollars³. ²https://espanol.cdc.gov/enes/flu/about/season/flu-season.htm³https://espanol.cdc.gov/enes/flu/about/disease/us_flu-related_deaths.htm

The most common diagnostic methods for the detection of Flu are called“Rapid Influenza Diagnostic Tests” (RIDT), these tests are based on thedetection of Flu antigens (immunoassay) in swab or nasopharyngealaspirate samples. These tests can give a result in a period of 15 to 20minutes, however, they lack sensitivity and only confer a qualitativeresult (positive or negative), which can potentially be a false negativedue to its low specificity⁴. ⁴https://espanol.cdc.gov/enes/flu/professionals/diagnosis/rapidlab.htm

Until now, the standard technique for confirming an influenza virusinfection corresponds to molecular analysis of reverse transcriptasepolymerase chain reaction (RT-PCR). For example, Human Influenza VirusReal-Time RT-PCR Diagnostic Panel developed by the Center for DiseaseControl and Prevention (CDC) enables in vitro detection of influenzavirus in respiratory tract samples from human patients exhibiting signsand symptoms of respiratory infection. This method detects influenza Aand B viruses through the reaction of primers against genes encodinghighly conserved proteins, such as matrix protein (M protein),nucleoprotein (NP protein) and non-structural protein (NS protein). Thistype of technique has disadvantages in terms of cost and optimization,since its implementation and start-up requires the acquisition ofhigh-cost specialized equipment and reagents, as well as highly trainedpersonnel.

Another common method for diagnosis is viral isolation in cell cultures.The problem with this type of technique is that it requires highlyspecialized equipment and personnel. On the other hand, it is a slowmethod that can deliver diagnostic results within 5 to 14 days after itsinitiation.

In the practice of clinical diagnosis, one of the main difficulties orproblems is the sample itself, since a limited amount of it can beaccessed, which also has a low antigen concentration and includes thepresence of other proteins and cellular components that can interferewith the detection reaction.

Monoclonal antibodies have been previously described for the detectionof human influenza virus antigens. In WO2012045001 (A2), for example, ahuman monoclonal antibody is disclosed that binds to the surface proteinhemagglutinin. In U.S. Pat. No. 9,650,434B2a monoclonal antibody orantigen-binding fragment thereof is provided, which can specificallybind to HA1 domain of the hemagglutinin protein of influenza viruses H1subtype and H5 subtype. In both documents, the proposed solution aims ata different antigen from the PB2 protein detection, and the efficiency,specificity and sensitivity of the antigen-antibody binding in clinicalsamples is not demonstrated.

Regarding detection of the Flu PB2 protein, JP2015189715 (A) provides amonoclonal antibody or an antigen-binding fragment thereof that binds tothe PB2 subunit of RNA-dependent RNA polymerase. Sequences of the heavychain variable regions and light chain variable regions of the antibodydescribed therein differ substantially from the monoclonal antibodies'sequences part of the invention. On the other hand, JP2015189715 (A)does not perform antigen detection assays in human clinical samples, northe specificity and sensitivity features of the antibodies isdetermined, in the context of clinical diagnosis. Let us remember thatin clinical diagnostic conditions, biological samples that include verylow concentrations of antigen are used, which hinders the specificityand sensitivity of the antigen-antibody reaction.

Therefore, a new alternative for the diagnosis of the human influenzavirus is required that, unlike molecular diagnostic tests and cellculture tests that entail longer response times and a high cost fortheir implementation and maintenance, allows the detection of a widevariety of influenza types and subtypes quickly, sensitively,specifically and at a lower cost. Furthermore, even though until nowmonoclonal antibodies have been proposed for detection of other Fluproteins and even against PB2, these antibodies have only been evaluatedin murine models and do not correspond in any case to a solution to theposed technical problem.

According to information provided, monoclonal antibodies that detect PB2protein are proposed to be used in detection and rapid, efficient andaccurate diagnosis in patients infected with Flu, where said antibodiesspecifically detect the protein in clinical samples at very lowconcentrations of the specific antigen (high sensitivity), evendistinguishing the specific viral antigen in clinical samples that eveninclude antigens from other respiratory viruses. Additionally, providedantibodies can form part of a diagnostic method and kit for the Fludiagnosis, where each antibody can be used in a versatile way as adetection antibody as well as a capture antibody.

DESCRIPTION OF THE INVENTION

The present invention relates to specific monoclonal antibodies againstPB2 protein fragments thereof, of the human influenza virus. Inparticular, the invention corresponds to monoclonal antibodies orfragments thereof secreted by hybridoma cell lines called 1A3E2 and2F11B1, that recognize the PB2 protein of the human influenza virus(Flu), where said monoclonal antibodies or fragments thereof comprise anantibody that comprises a light chain variable region where its CDR1(CDR_(LC1)) is defined according to SEQ ID NO: 1, its CDR2 (CDR_(LC2))is defined by SEQ ID NO: 2 and its CDR3 (CDR_(LC3)) corresponds to SEQID NO: 3, and a heavy chain variable region where its CDR1 (CDR_(HC1))is defined according to SEQ ID NO: 4, its CDR2 (CDR_(HC2)) is defined bySEQ ID NO: 5 and its CDR3 (CDR_(HC3)) corresponds to SEQ ID NO: 6, or anantibody comprising a light chain variable region where its CDR1(CDR_(LC1)) is defined according to SEQ ID NO: 7, its CDR2 (CDR_(LC2))is defined by SEQ ID NO: 8 and its CDR3 (CDR_(LC3)) corresponds to SEQID NO: 9, and a heavy chain variable region where its CDR1 (CDR_(HC1))is defined according to SEQ ID NO: 10, its CDR2 (CDR_(HC2)) correspondsto SEQ ID NO: 11 and its CDR3 (CDR_(HC3)) corresponds to SEQ ID NO: 12,where said antibody can be used as detection or capture antibody.Additionally, a method for diagnosing Flu infection in a biologicalsample is provided that uses monoclonal antibodies in diagnostic kitformat to detect Flu, where said kit comprises at least one monoclonalantibody against Flu as previously described.

Antibodies described in the invention have important advantageous andremarkable technical features with respect to other antibodies andmethods for detecting viral antigens already existing.

First, each virus has specific surface proteins, therefore, otherdiagnostic techniques based on monoclonal antibodies for other types ofrespiratory viruses are not comparable to the proposed invention.Detection specificity of antibodies against Flu PB2 protein respect toother viral antigens, for example against adenovirus pill protein, isdemonstrated in the results provided in FIGS. 1A, 1B and 1C of thepresent application. From these results it is possible to conclude thatthe provided antibodies in the scope of the present application onlyrecognize the Flu PB2 protein, and that in ELISA assays with ADV virusantigens no detection signal was observed.

Second, antibodies that are part of the scope of the invention allowspecific detection of PB2 protein fragments thereof, in such a way thatthey do not compete with each other for the antigen-binding site, nor dothey exert an impediment to simultaneously bind to it.

Third, they allow the detection of PB2 protein or fragments thereof withhigh sensitivity in samples containing a small antigen amount, such asnasopharyngeal swab samples, for example.

The proposed monoclonal antibodies are capable of detecting PB2 protein,a highly conserved protein. Strategy for detecting a conserved viralprotein allows antibodies that are part of the scope of the invention todetect different types of human influenza, including influenza A, B andC.

When reference is made to CDR sequences in the present invention, thesecorrespond to short sequences found in the variable domains of proteinsthat have antigen detection function. CDR sequences for the heavy chain(CDR_(HC)) and light chain (CDR_(LC)) of the antibodies secreted byhybridomas 1A3E2 and 2F11B1.

Monoclonal antibodies described can be used for Flu infectiondetermination, diagnosis and/or detection assays. These antibodies canbe used simultaneously to increase detection sensitivity of clinicalsamples where there is little quantity and availability of antigen. Inthis regard, it is also provided a Flu infection diagnosis method in abiological sample, which comprises contacting the biological sample withthe monoclonal antibody against Flu PB2 protein or a fragment thereofaccording to the claim, and detecting the binding of the antibody withthe antigen. Biological sample can be, but is not limited to, infectedin vitro cells with Flu, nasal secretions, nasal washes, cerebrospinalfluid, pharyngeal secretions and/or bronchial washes or secretions. Aspart of the method, the assay used for the detection of antigen-antibodybinding is selected from ELISA, luminex, immunofluorescence,immunohistochemistry, immunochromatography, flow cytometry, cell sorter,immunoprecipitation and/or Western blot.

Present invention also includes a diagnostic kit to detect the humaninfluenza virus, which comprises: a monoclonal antibody against Flu PB2protein or a fragment thereof, where said antibody can act as a captureor detection antibody, where particularly, the detection antibody isconjugated to a marker for its detection; a solid support to which theantibody is attached; and reagents for detecting the marker included inthe detection antibody, such as fluorophores, biotin, radioisotopes,metals, and enzymes.

In the present invention when it refers to capture antibody, thiscorresponds to the antibody that specifically binds to the antigen. Inthe case of the detection antibody, this corresponds to the antibody towhich a marker is conjugated to be detected by different tests such asimmunochromatographic test, luminex, flow cytometry, immunofluorescence,radioimmunoassay, Western blot, Dot plot, ELISA, luminex,immunodiffusion. or immunoprecipitation.

Antibodies that are part of the present invention can act dually as acapture antibody or as a detection antibody when coupled to a detectionmarker. Detection marker will be conjugated to the detection antibody,and this can correspond, without limitation, to fluorophores, biotin,radioisotopes, metals and enzymes. Preferably, the detection antibody isconjugated to reporter system based on the detection of horseradishperoxidase (HRP) enzyme activity.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: Flu PB2 protein detection by monoclonal antibodies produced by1A3E2 and 2F11B1 hybridomas, using an indirect ELISA assay. The platewas activated with 50 ng of purified Flu recombinant PB2 protein, 50 ngof ADV pIII protein (as a specificity control) and 20 μg of uninfected(used as a specificity control) and Flu-infected MDCK cells. Controlwells with no antigen, with primary antibody, with HRP-conjugatedanti-mouse IgG (not activated) and wells with no antigen or primaryantibody, only with anti-mouse IgG (HRP), data not shown in the graph.Subsequently, wells were incubated with the anti-PB2 antibodies from the1A3E2 hybridoma, in an amount of 170 ng (A), 2F11B1 hybridoma in anamount of 170 ng (B) and the commercial polyclonal antibodyAnti-Influenza A virus PB2 protein antibody, catalog number GTX125926(GeneTex) used in an amount of 170 ng (C). Data shown in the graphexpress detected absorbance (in OD, optical density) at 450 nm, emittedby the conversion of Tetramethylbenzidine substrate to a coloredcompound, catalyzed by Horseradish peroxidase (HRP)enzyme conjugated ina secondary anti-mouse IgG antibody that specifically bound antibodiessecreted by GeneTex hybridomas 1A3E2, 4D8C6 and GTX125926 hybridomas.Values correspond to the standard deviation in absorbanceaverage+/−emitted by each sample in at least two independentexperiments. Where, *P<0.05; **P<0.01; ***P<0.001 and ****P<0.0001 byparametric student test comparing the results of the pIII-ADV proteinversus those of PB2-Flu, and on the other hand comparing the uninfectedversus infected cells.

FIG. 2: Sensitivity determination of monoclonal antibodies produced by1A3E2 and 2F11B1 hybridomas in the detection of Flu PB2. ELISA plateswere activated with 1:2 serial dilutions, starting with 50 ng of PB2protein ending with 0.04 ng. Subsequently, the wells were incubated withthe anti-PB2 antibodies from the 1A3E2 hybridoma, in an amount of 170 ng(A) and the 2F11B1 hybridoma in an amount of 170 ng (B). Non-activatedwells were included as a negative control. Data shown in the graphexpress absorbance at 450 nm, emitted by the conversion ofTetramethylbenzidine substrate to a colored compound, catalyzed byHorseradish peroxidase (HRP) enzyme conjugated to anti-PB2 antibodiesfrom 1A3E2 and 2F11B1 hybridomas in an amount of 170 ng (A and B).Values correspond to the standard deviation in absorbanceaverage+/−emitted by each sample in at least two independentexperiments. *P<0.05; **P<0.01 and ***P<0.001 by parametric student testcomparing the results of well called control versus each of thedilutions of PB2 protein.

FIG. 3: Assay of serial dilutions of Flu anti-PB2 monoclonal antibodiesproduced by 1A3E2 and 2F11B1 hybridomas, for the detection of purifiedFlu antigens. ELISA plates were activated with 50 ng of Flu recombinantPB2 protein and antigen was detected with 11 serial dilutions ofanti-1A3E2 PB2 antibodies (A) or 2F11B1 (B) 1:2, starting from aconcentration of 3.4 μg/mL (170 ng per well). Values are expressed asstandard deviation average+/−of the value of absorbance emitted at 450nm of each duplicate sample, in at least two independent experiments.*P<0.05; **P<0.01 and ***P<0.001 by parametric student test comparingthe results of well called control versus each dilution of PB2 protein.

FIG. 4: Flu detection in clinical samples by ELISA Sandwich, using thecombination of monoclonal antibodies secreted by 1A3E2 and 2F11B1hybridomas. ELISA plates were activated with 170 ng of secreted antibodyby 1A3E2 hybridoma (anti-Flu), functioning as capture antibody.Activated wells with capture antibody were incubated with 50 μL ofnasopharyngeal swab (NPS) samples from patients with viral respiratorysymptoms. As negative controls, 10 samples of healthy controls wereanalyzed. 12 samples of patients Flu-positive were used and as aspecificity control, 3 samples of parainfluenza virus positive patientswere included. As a positive control, wells were included to whichpurified Flu recombinant PB2 protein was added. For the detection ofcaptured protein by 1A3E2 antibody, antibodies produced by 2F11B1hybridoma, conjugated to the Horseradish Peroxidase enzyme, were used ina 1:2000 dilution (1.8 ng/μL per well). Data shown the median value ofemitted absorbance at 450 nm of each sample (**P<0.01 and ****P<0.0001;using the non-parametric student test and Mann Whitney post testcomparing Flu positive patients versus healthy controls, and againstviruses used as specificity control).

FIG. 5: Protein PB2 detection by indirect ELISA, using monoclonalantibodies secreted by biotin-conjugated 1A3E2 and 2F11B1 hybridomas.PB2 protein detection of Biotin-conjugated antibodies is observed.Antibody fragments secreted by 1A3E2 and 2F11B1 hybridomas are indicatedin black and white respectively. While activity of the completefragments of antibodies secreted by 1A3E2 and 2F11B1 hybridomasrespectively is shown in gray. Data shown in graph express absorbance at450 nm emitted by the conversion of substrate Tetramethylbenzidine to acolored compound catalyzed by the Horseradish peroxidase (HRP) enzyme.Average value of emitted absorbance at 450 nm of each sample is shown(where b is equal to p<0.0001 compared to a; by means of the 2-way ANOVAtest comparing the well with no sample versus well with protein with allantibodies).

EXAMPLES THAT MAKE IT POSSIBLE TO DEMONSTRATE THE DIFFERENT APPLICATIONSOF THE MONOCLONAL ANTIBODIES OF THE INVENTION Example 1: Determinationof the Nucleotide Sequence Encoding the Light (VL) and Heavy (VH) Chainsof the Variable Region of Flu Anti-PB2 Antibody Secreted by 1A3E2Hybridoma

1A3E2 hybridoma was grown in DMEM-high glucose culture mediumsupplemented with 3.7 g/L of Sodium Bicarbonate and 10% fetal bovineserum, at 37° C. (98.6° F.) with 10% CO₂, up to a cell density of700,000 cells/mL. Total RNA of 3.5×10⁶ cells was obtained, performing atreatment with Trizol compound (Invitrogen). 0.5 μg of RNA was used togenerate the cDNA by reverse transcription reaction with thePrimeScript™ 1st Strand cDNA Synthesis kit, which uses isotype-specificuniversal primers. The antibody heavy and light chain were amplifiedaccording to the GenScript rapid amplification of cDNA ends (RACE)standard operating procedure (SOP). Amplified antibody fragments wereseparately cloned into a standard cloning vector. PCR colony wasperformed to identify clones which have the correct size inserts. Atleast five colonies with inserts of the correct size were sequenced foreach fragment. Sequences of different clones were aligned and theconsensus sequence of these clones was provided. Nucleotide sequences ofheavy and light chains of antibodies secreted by 1A3E2 hybridoma wereidentified, being identified as SEQ ID NO. 1 and SEQ ID NO.3 for thecase of heavy chains and SEQ ID NO. 2 and SEQ ID NO.4 for the case oflight chains.

Example 2: Determination of the Nucleotide Sequence Encoding the Light(VL) and Heavy (VH) Chains of the Variable Region of Flu Anti-PB2Antibody Secreted by 2F11B1 Hybridoma

2F11B1 hybridoma was grown in DMEM-high glucose culture mediumsupplemented with 3.7 g/L of Sodium Bicarbonate and 10% fetal bovineserum, at 37° C. (98.6° F.) with 10% CO₂, up to a cell density of700,000 cells/mL. Total RNA of 3.5×10⁶ cells was obtained, performing atreatment with Trizol compound (Invitrogen). 0.5 μg of RNA was used togenerate the cDNA by reverse transcription reaction with thePrimeScript™ 1st Strand cDNA Synthesis kit, which uses isotype-specificuniversal primers. The antibody heavy and light chain were amplifiedaccording to the GenScript rapid amplification of cDNA ends (RACE)standard operating procedure (SOP). Amplified antibody fragments wereseparately cloned into a standard cloning vector. PCR colony wasperformed to identify clones which have the correct size inserts. Atleast five colonies with inserts of the correct size were sequenced foreach fragment. Sequences of different clones were aligned and theconsensus sequence of these clones was provided. From this, nucleotidesequences of heavy and light chains of antibodies secreted by 2F11B1hybridoma were determined, corresponding to those identified as SEQ IDNO. 1 and SEQ ID NO.3 to the light chains and sequences identified asSEQ ID NO. 1 and SEQ ID NO.3 to heavy chains.

Example 3: Flu Antigen Detection Assay, Specificity Determination of FluAnti-PB2 Monoclonal Antibodies for Purified Flu Antigens by IndirectELISA Assay

This assay aims to demonstrate the specificity for Flu PB2 proteinantibodies produced by 1A3E2 and 2F11B1 hybridomas. Antigen detectionwas carried out using the indirect ELISA technique, where ELISA platewas activated with 50 ng of purified antigen for 1 hour at 37° C. (98.6°F.). Similarly, the plate was activated with 20 μg of uninfected MDCKcell lysate (as a negative control) and infected with Flu serotype Avirus. Another negative control included was 50 ng of ADV pIII proteinin a separate well. Subsequently, the plate was washed twice with1×/Tween20 0.05% phosphate buffered saline (PBS). The plate was thenblocked for 2 hours at 37° C. (98.6° F.) with 1×PBS/10% Fetal BovineSerum (FBS). Subsequently, the washes were repeated and then eachantibody (1A3E2 and 2F11B1) were incubated at a final concentration of3.4 μg/mL (170 ng per well), diluted in 1×PBS/10% FBS, for 1 hour at 37°C. (98.6° F.) (each antibody on a separate plate). Under the sameconditions, on a different plate, a control assay was performed using acommercial monoclonal antibody that recognizes the PB2 protein of Flu(Anti-Influenza A virus PB2 protein antibody, catalog number GTX125926,GeneTex) at a concentration of 3.4 μg/mL. After incubation time, thewashes were repeated and a secondary anti-mouse IgG antibody labeledwith horseradish peroxidase (HRP) in dilution 1 in 2000 (1.8 ng/μl perwell) was added to each well in 1×PBS/10% FBS, for 1 hour at roomtemperature (≈25° C. (77° F.)), in the dark. Finally, washes werecarried out and it was developed with 50 μL ofcitrate/tetramethyl-benzidine buffer (TMB,3,3′,5,5′-tetramethylbenzidine, 1 mg/mL, Becton Dickinson). To stop thereaction, 50 μL of H₂SO₄ 2 N were added and the result was read on anELISA reader, at 450 nm. To determine that the reaction of the secondaryantibody was specific in recognizing the primary antibody and also thatthe obtained signal was not caused by nonspecific binding of thesecondary antibody to the viral antigen, controls were carried out inwhich only the secondary antibody was used with no primary antibody orsample (well not activated). Another control to determine that theprimary antibody reaction is specific for the antigen, consisted ofusing the antibodies on an ELISA plate that has not been activated withthe antigen (with no antigen) or using the antibodies on an ELISA platethat possessed 50 ng of ADV pIII protein or uninfected cells. Resultsshow that monoclonal antibodies of the invention are capable ofrecognizing 50 ng of purified antigen, specifically, since they do notrecognize ADV pIII protein, nor proteins of uninfected cells (FIGS. 1Aand 1B). On the other hand, it was observed that commercial antibody(FIG. 1C) used in the assay as a control, although it was specific forthe detection of infected cells only, it was not efficient in detectingpurified Flu recombinant PB2 protein in our laboratory. All negativecontrols used gave expected results (data not shown in the figures).

Example 4: Assay to Determine Monoclonal Antibodies Sensitivity for theDetection of Flu Anti-PB2 Viral Antigens

Assay was performed to determine the maximum protein dilution that Fluanti-PB2 monoclonal antibodies from 1A3E2 and 2F11B1 hybridomas are ableto detect by indirect ELISA. For this, the same technique described inexample 3 was used. The plate was activated with 11 serial dilutions ofFlu PB2 protein 1:2, starting with 50 ng of purified antigen. Anti-PB21A3E2 and 2F11B1 antibodies were used in a concentration of 3.4 μg/mL(170 ng/well), and were diluted in 1×PBS/10% FBS. Subsequently,anti-mouse IgG detection antibody was added in a dilution of 1:2,000(1.8 ng/μL per well) and incubated for 1 hour at room temperature (≈25°C. (77° F.)), in the dark. Finally, the washes were carried out and itwas developed with 50 μL of citrate/Tetramethylbenzidine (TMB,3-3′-5-5′-tetramethylbenzidine, 1 mg/mL, Becton Dickinson) buffer. Tostop the reaction, 50 μL of H₂SO₄ 2 N were added and the result was readon an ELISA reader, at 450 nm. Results showed that anti-PB2 1A3E2antibody is capable of detecting up to 780 picograms (pg) of the Flu PB2protein (FIG. 2A). Anti-PB2 antibody from 2F11B1 hybridoma showed thesame sensitivity as anti-PB2 1A3E2 antibody (FIG. 2B). Controls wereincluded in all the tests which allowed to rule out non-specificreactions of both the antibodies, which contained all components of thetest except the sample (Flu PB2 protein, data not shown in the graphs).

Example 5: Assay to Determine Monoclonal Antibodies Efficiency to DetectFlu Viral Antigens, by Indirect ELISA

Assay was performed to determine the maximum dilution of Flu anti-PB2monoclonal antibodies from 1A3E2 and 2F11B1 hybridomas which allow thedetection of the viral antigen. For this, a plate was activated with 50ng of purified antigen (protein PB2) and then the plate was blocked for2 hours at 37° C. (98.6° F.) with 1×PBS/10% Fetal Bovine Serum (FBS).Anti-PB2 1A3E2 and 2F11B1 antibodies were used in 1:2 dilutions,starting from the working concentration (170 ng) up to dilution 11 (0.15ng) in 1×PBS/10% FBS. Subsequently, anti-mouse IgG detection antibodywas added in a dilution of 1:2000 (1.8 ng/μL per well) incubated for 1hour at room temperature (≈25° C. (77° F.)), in the dark. Finally, thewashes were carried out and it was developed with 50 μL ofcitrate/Tetramethylbenzidine (TMB, 3-3′-5-5′-tetramethylbenzidine, 1mg/mL, Becton Dickinson) buffer. To stop the reaction, 50 μL of H₂SO₄ 2N were added and the result was read on an ELISA reader, at 450 nm. InFIG. 3 is observed that anti-PB2 1A3E2 antibody can detect 50 ng of thepurified antigen up to 1.3 ng per well (FIG. 3A). On the other hand, theanti-PB2 2F11B1 clone is more efficient than the 1A3E2 clone, since itrecognizes 50 ng of purified PB2 with almost all the dilutions made(FIG. 3B). Negative control included in this assay corresponds to a wellwhich does not contain sample (protein PB2), was blocked with 1×PBS/10%FBS, primary antibody (anti-PB2 1A3E2 or anti-PB2 2F11B1) was added andalso contains HRP-conjugated anti-mouse IgG antibody.

Example 6: Clinical Diagnosis of Samples from Flu-Infected Patients,Using Flu Anti-PB2 Monoclonal Antibodies, Using ELISA Sandwich Technique

Availability and concentration of viral proteins is generally very lowin clinical samples of nasopharyngeal swabs, so it was necessary tomodify the ELISA assay that was previously performed. For this assay, aSandwich ELISA was performed, using anti-PB2 antibody from the Flu 1A3E2hybridoma as capture antibody and Flu anti-PB2 2F11B1 clone as detectionantibody. Flu anti-PB2 2F11B1 detection antibody was conjugated to theHRP. Wells of an ELISA plate were activated with 3.4 μg/mL (170 ng/well)of anti-PB2 antibody from Flu 1A3E2 hybridoma, diluted in 1×PBS, for 1hour at 37° C. (98.6° F.). 2 washes were carried out with 1×-Tween20 PBS0.05% and later the plate was blocked with 200 μL of 1×PBS/10% FBS for 1hour at 37° C. (98.6° F.). Washed again and incubated for 1 hour at 37°C. (98.6° F.) each well with 50 μL of nasopharyngeal swabs (previouslytreated) from patients positive for Flu according to the diagnosticmethod “D³ Ultra DFA Respiratory Virus Screening and ID ( ) Kit de DHI(Diagnostics Hibryds) USA”, routinely referred to as “viral panel”, andwhich were treated as described later. As controls were included: 1)specificity control: 50 μL of sample of patients diagnosed with Flu wereused by the viral panel for anti-Flu antibodies; 2) positive control: 50ng of recombinant PB2-Flu protein; 3) Negative control: corresponding tohealthy control samples. Subsequently, the 2 corresponding washes werecarried out with 1×-Tween20 PBS 0.05% and each well was incubated for 1hour at room temperature with 50 μl of anti-PB2 antibody from 2F11B1hybridoma, conjugated with HRP (1.8 ng/μL of final concentration).Detection antibodies were incubated for 1 hour at room temperature (≈25°C. (≈77° F.)), in the dark. The plate was then washed 2 more times,developed with 50 μL of TMB solution and incubated for 15 minutes in thedark. The reaction stopped with 50 μL of H2SO₄ 2N and the plate was readat 450 nm in an ELISA reader (Epoch model), certified for clinicaldiagnosis.

Obtained results for this test are shown in FIG. 4A, where it can beobserved that the ELISA Sandwich technique using the antibody (anti PB2)from 1A3E2 hybridoma, as capture antibody and the antibody from the2F11B1-HRP hybridoma as detection antibody, allows the detection of theantigen in samples of Flu-infected patients (FIG. 4A), which werepreviously confirmed by direct immunofluorescence in a certifiedclinical laboratory using the viral panel. FIG. 4A, shows the obtainedresults with Flu anti-PB2 antibodies, where 12 samples from patientsdiagnosed as positive PIV were used and as a specificity control, 3samples from patients positive for the Influenza virus were included. Asa positive control, wells were included to which purified Flurecombinant PB2 protein was added. As negative control, 10 healthycontrols were analyzed. Results show that antibodies are specific indetecting only Flu-positive patients and not healthy controls or thoseinfected with another virus (PIV). All samples detected positive byELISA are those that show an optical density (OD) above 0.15.

This assay demonstrates the versatility of the antibodies from 1A3E2 and2F11B1 hybridomas Flu anti-PB2, since they are capable of simultaneouslybinding to the antigen without competing for the binding site orinterfering with each other. The above allows the capture and subsequentdetection of PB2 protein in patient samples.

Treatment of clinical samples. The samples used for the tests wereobtained from nasopharyngeal swabs contained in universal transportmedium (UTM). The samples were centrifuged at 14,000 rpm for 4 minutesat room temperature. Subsequently, the supernatant (SN1) was separatedfrom the pellet; the latter was incubated with 100 μL of RIPA Buffer (50mM Tris-HCl pH 8.0, 150 mM NaCl, 1% NP-40, 0.5% Sodium Deoxycholate,0.1%, SDS and a 1× protease inhibitor cocktail) for 15 minutes at 4° C.(39.2° F.), vortexing every 5 minutes. It was then centrifuged at 14,000rpm for 4 minutes at room temperature. At the end, the supernatantobtained (SN2) was taken and mixed with SN1, vortexing was performed.

It is extremely important to use both antibodies for the detection ofPB2 protein, due to the low availability of antigen in the sample. Usingan ELISA Sandwich increases the specificity and sensitivity in thediagnosis of Flu. Assays were performed where the plate was activateddirectly with clinical samples of nasopharyngeal swabs, then anti-PB21A3E2 and anti-PB2 2F11B1 antibodies were incubated, separately. Then asecondary anti-mouse IgG antibody conjugated with HRP was incubated andabsorbance generated by incubating the antibody complex plus sample withthe TMB substrate was evaluated, and a positive diagnosis was notobserved since the signal delivered was very low (data not shown).

Carrying out a diagnostic kit using the ELISA's Sandwich technique,where the plate can be activated and blocked, would reduce the time andcost of performing a diagnosis, since this technique is easy to performand analyze compared to the standard technique (PCR). The kit does notneed highly trained personnel to perform or analyze it.

Example 7: Clinical Diagnosis of Samples from FLU-Infected Patients,Using FLU Anti PB2 Monoclonal Antibodies, by Luminex Sandwich-Type

As in ELISA technique, the availability and concentration of viralproteins is generally very low in clinical samples of nasopharyngealswabs, so it was wanted to evaluate the obtained results by ELISAtechnique to another more sensitive technique (FIG. 4A). For this assay,a Sandwich-type luminex assay was performed, using anti-PB2 1A3E2antibody as capture antibody and anti-PB2 2F11B1 as detection antibody.FLU anti-PB2 2F11B1 detection antibody was conjugated to the fluorophorebiotin. Luminex plates were activated with 50 magnetic microspheres per(internally labeled with red or near infrared fluorophore of differentintensities) per μL, which were conjugated with the antibody secreted by1A3E2 hybridoma (anti-FLU), functioning as a capture antibody (at afinal concentration of 2.5 μM). Conjugated microspheres were incubatedwith 50 μL of nasopharyngeal swab (NPS) samples from patients with viralrespiratory symptoms, for 2 hours at room temperature (□23° C. (□73.4°F.)), stirring at 400 rpm and in the dark (covered with aluminum foil).As negative controls, 8 samples of healthy controls were analyzed. 19samples of patients positive for Flu were used (according to thediagnostic method “D³ Ultra DFA Respiratory Virus Screening and ID Kitde DHI (Diagnostics Hibryds) USA”, routinely referred to as “viralpanel”, which were treated as the same way mentioned above, and as apositive control, wells were included to which purified PB2-FLU protein(50 ng) was added. After 2 hours, 2 washes are carried out again with100 μL 1×-Tween20 PBS 0.05% for 30 seconds using the manual magneticscrubber. For detection of protein captured by 1A3E2 antibody,antibodies produced by 2F11B1 hybridoma, conjugated to biotinfluorophore, were used at a concentration of 4 μg/mL diluted in 1×PBS-1%BSA, the wells being incubated with 50 μL. Incubation is carried out for1 hour at room temperature, in the dark, stirring at 400 rpm. 2 washesare carried out again with 100 μL 1×-Tween20 PBS 0.05% for 30 secondsusing the manual magnetic scrubber. The complex formed by conjugatedmicrospheres with capture antibody plus antigen and detection antibodyis incubated with 50 μL of Streptavidin/Phycoerythrin at a finalconcentration of 6 μg/mL. Incubation is carried out for 30 minutes atroom temperature, in the dark, stirring at 400 rpm. Finally, two morewashing steps are carried out and the wells are incubated with 100 μL ofSheat fluid reagent (reagent used by Luminex equipment for the equipmentto read the samples), stir 5 minutes at 400 rpm, in the dark. Results ofthe mean fluorescence intensity (MFI) are then read on the Luminex 200equipment, which, through a red laser (621 nm), detects the recognitionregion of the microsphere and the Green laser (511 nm) detects thebinding of the detection antibody to the analyte.

Obtained results for this test are shown in FIG. 4B, where it can beobserved that the Luminex technique, as the obtained by ELISA techniqueusing the antibody (anti PB2) from 1A3E2 hybridoma, as capture antibodyand the antibody from the 2F11B1-HRP hybridoma as detection antibody,allows the detection of the antigen in samples of FLU-infected patients(FIG. 4B) with high intensity, which were previously confirmed by directimmunofluorescence in a certified clinical laboratory using the viralpanel. FIG. 4B, shows the obtained results with FLU anti-PB2 antibodies,where 19 samples from patients diagnosed as positive FLU were used and 6healthy control samples. Furthermore, as a positive control, wells wereused to which purified PB2-FLU protein was added. Results show thatanti-PB2 antibodies are specific in detecting only Flu-positive patientsand not control subjects. All samples detected as positive by Luminexare those that show an MFI above two standard deviations from the meanMFI of healthy controls.

This assay, as in ELISA assay with patient samples, demonstrates theversatility of antibodies from 1A3E2 and 2F11B1 hybridomas of FLU, sincethey are capable of simultaneously binding to antigen without competingfor the binding site or interfere with each other and detect poorantigen availability in nasopharyngeal swab sample.

Example 8: Blind Study for the Detection of PB2-FLU Antigen in ClinicalSamples, Obtained from Patients with an Infection, Using Flu Anti-PB2Monoclonal Antibodies, which are Part of the Respiratory Virus MultipleDetection Kit

Previously, ELISA tests were carried out in Sandwich where the previousdiagnosis of the samples to be evaluated was known. After these tests, ablind study was carried out, where about 160 nasopharyngeal swab sampleswere evaluated, without knowing the microbiological diagnosis. For allassays in the blinded study, ELISA's Sandwich were performed whereanti-L 1A3E2 antibody was used as capture antibody and anti-L 2F11B1antibody was used as HRP-conjugated detection antibody. For all assays,wells of an ELISA plate were activated with 3.4 μg/mL (170 ng/well) ofanti-L antibody from FLU 1A3E2 hybridoma, diluted in 1×PBS, for 30minutes at 37° C. (98.6° F.). 2 washes were carried out with 1×-Tween20PBS 0.05% and later the plate was blocked with 200 μL of 1×PBS/10% FBSfor 30 minutes at 37° C. (98.6° F.). Each well with 50 μL ofnasopharyngeal swabs from patients was washed again and incubated for 1hour at 37° C. (98.6° F.), which were evaluated in parallel by thestandard diagnostic method (PCR), routinely referred to as “viralpanel”, and which were treated as previously described in example 6. Ascontrols were included: 1) specificity control: 50 μL of BSA protein (50ng) were used; 2) positive control: 50 ng of PB2-FLU recombinantprotein; 3) Negative controls: wells with no sample and wells blockedand incubated with detection antibody. Subsequently, the 2 correspondingwashes were carried out with 1×-Tween20 PBS 0.05% and each well wasincubated for 30 minutes at room temperature (≈25° C. (≈77° F.), in thedark) with 50 μl of anti-PB2 antibody from 2F11B1 hybridoma, conjugatedwith HRP (1.8 ng/μL of final concentration). The plate was then washed 2more times, developed with 50 μL of TMB solution and incubated for 15minutes in the dark. The reaction stopped with 50 μL of H2SO₄ 2 N andthe plate was read at 450 nm in an ELISA reader (Epoch model), certifiedfor clinical diagnosis.

Results are shown in FIG. 4A, where the ability of antibodies to detectprotein PB2 in clinical samples is observed, since they were designedfrom a chimera protein. 18 out of 21 PIV positive patients weredetected, and from these results the diagnostic accuracy of antibodiescould be determined, which is shown in Table 1. The table shows the twoconcepts that define diagnostic accuracy, where we have specificity,that is, the ability of antibodies to diagnose negative samples asnegative, without detecting false positives, and on the other hand, wehave sensitivity, that is, the ability of antibodies to diagnose aspositive those samples that really are, without diagnosing falsenegatives. Exposed results in the table show a high specificity (94%)and sensitivity (86%) percentage of antibodies against the standardtechnique (PCR).

TABLE 1 Diagnostic accuracy of anti-PB2-FLU antibodies PIV (N =Diagnosis by reference 160) technique: PCB Specificity SensitivityDiagnostic True positives False positives 100% 86% test: ELISA 18 9False negatives True negatives 3 130

Example 9: Protein PB2 Detection by Indirect ELISA Assay, Using CompleteMonoclonal Antibodies or Fragments Thereof

In this application example, is demonstrated that both the specificmonoclonal antibody against the PB2 protein can be detected by indirectELISA. For detection of protein L, ELISA plates were activated with 50μL of protein PB2 y BSA 50 ng. Nonspecific sites were blocked with 10%FBS diluted in 1×PBS. 170 ng (3.4 μg/mL) of Fab fragments of antibodiessecreted by 1A3E2 (anti-Flu) and 2F11B1 (anti-Flu) hybridomas, bothpreviously biotin conjugated. Incubation of biotin-binding molecules(Streptavidin), which is HRP-conjugated (1:2,000 dilution, 75 ng perwell) (FIG. 5, dark gray bar, 1A3E2 antibody and light gray bar, 2F11B1antibody).

Example 10: Flu Antigen Detection Assay, Using F(Ab′)2 Fragments of FluAnti-PB2 Monoclonal Antibodies by Indirect ELISA

The objective of this assay is to demonstrate the ability to detectfragments of anti-Flu antibodies, produced by 1A3E2 and 2F11B1hybridomas, by PB2 protein. Prior to the indirect ELISA assay, IgGmolecule of each anti-Flu antibody was fragmented. Fragmentation wasperformed using the “Thermo Scientific™ Pierce™ F(ab′)₂ FragmentPreparation Kits” kit (#10381214, Thermo Scientific), which separatesF(ab′)₂ fragment and Fc from the antibody of interest, by using theenzyme pepsin that digests the Fc fragment and subsequently purificationsteps are carried out to separate the F(ab′)₂ fragment from the digestedFc fragment. After antibody fragmentation, purified F(ab′)₂ fraction wasverified by the Western bolt technique. F(ab′)₂ fractions wereconjugated to biotin molecules using the rapid conjugation kit,Lightning-Link rapid biotin type A (#370-0010, Expedeon). Having readyall reagents, antigen detection was carried out by indirect ELISAtechnique, where ELISA plate was activated with 50 ng of purified PB2antigen for 1 hour at 37° C. (98.6° F.). Two negative controls wereincluded, one with no sample and the other incubating the well with 50ng of BSA protein. Subsequently, the plate was washed twice with1×/Tween20 0.05% phosphate buffered saline (PBS). The plate was thenblocked for 2 hours at 37° C. (98.6° F.) with 1×PBS/10% Fetal BovineSerum (FBS). Subsequently, the washes were repeated and then eachantibody conjugated with biotin, unfractionated and F(ab′)₂ fractions(1A3E2 and 2F11B1) was incubated at a final concentration of 3.4 μg/mL(170 ng per well), diluted in 1×PBS/10% FBS, for 1 hour at 37° C. (98.6°F.) (each antibody on a separate plate). After incubation time, washingswere repeated and a biotin-binding protein (Streptavidin) labeled withhorseradish peroxidase (HRP) enzyme was added to each well in dilution 1in 2000 (25 ng/μL per well) in 1×PBS/10% FBS, for 1 hour at roomtemperature (□25° C. (□77° F.), in the dark. Finally, the washes werecarried out and it was developed with 50 μL ofcitrate/Tetramethylbenzidine (TMB, 3-3′-5-5′-tetramethylbenzidine, 1mg/mL, Becton Dickinson) buffer. To stop the reaction, 50 μL of H₂SO₄ 2N were added and the result was read on an ELISA reader, at 450 nm. Todetermine that the reaction of the secondary antibody was specific inrecognizing the primary antibody and also that the obtained signal wasnot caused by nonspecific binding of the secondary antibody to theantigen, controls were carried out in which only the secondary antibodywas used with no primary antibody or sample (well not activated).Another control to determine that the primary antibody reaction isspecific for the antigen, consisted of using the antibodies on an ELISAplate that has not been activated with the antigen (with no sample) orusing the antibodies on an ELISA plate that possessed 50 ng of PB2.Results show that monoclonal antibodies of the invention are capable ofrecognizing 50 ng of purified antigen, specifically, regardless ofwhether the complete antibody or a fragment thereof is used (FIG. 5,black bar, 1A3E2 antibody and white bar, 2F11B1 antibody).

1. A monoclonal antibody or an antigen-binding portion thereof thatbinds to human influenza virus (FLU) protein PB2 for use in detectingthe presence and/or localization of the protein, wherein the antibody isselected from: i) an antibody comprising a light chain variable regionwhere its CDR1 (CDR_(LC1)) is defined according to SEQ ID NO: 1, itsCDR2 (CDR_(LC2)) is defined by SEQ ID NO: 2 and its CDR3 (CDR_(LC3))corresponds to SEQ ID NO: 3, and a heavy chain variable region where itsCDR1 (CDR_(HC1)) is defined according to SEQ ID NO: 4, its CDR2(CDR_(HC2)) is defined by SEQ ID NO: 5 and its CDR3 (CDR_(HC3))corresponds to SEQ ID NO: 6, or ii) an antibody comprising a light chainvariable region where its CDR1 (CDR_(LC1)) is defined according to SEQID NO: 7, its CDR2 (CDR_(LC2)) is defined by SEQ ID NO: 8 and its CDR3(CDR_(LC3)) corresponds to SEQ ID NO: 9, and a heavy chain variableregion where its CDR1 (CDR_(HC1)) is defined according to SEQ ID NO: 10,its CDR2 (CDR_(HC2)) corresponds to SEQ ID NO: 11 and its CDR3(CDR_(HC3)) corresponds to SEQ ID NO: 12, wherein said antibody can beused as detection or capture antibody.
 2. A method to detect Flu virusin a biological sample comprising contacting the biological sample withthe monoclonal antibody or an antigen-binding portion thereof that bindsto Flu PB2 protein of claim 1 and detecting the binding of the antibodyto antigen, thereby detecting the Flu virus in the sample.
 3. The methodto detect Flu virus in a biological sample according to claim 2, whereinthe biological sample is selected from the group consisting of in vitrocells infected with Flu, nasal secretions, nasal washes, cerebrospinalfluid, pharyngeal secretions and/or bronchial washes or secretions. 4.The method to detect Flu virus in a biological sample according to claim2, wherein an assay used to detect the binding of the antibody toantigen is selected from: ELISA, immunofluorescence,immunohistochemistry, immunochromatography, flow cytometry, cell sorter,immunoprecipitation and/or Western blot.
 5. The method to detect Fluvirus in a biological sample according to claim 2, wherein the antibodyor an antigen-binding portion thereof is conjugated with a marker thatallows its detection.
 6. The method to detect Flu virus in a biologicalsample according to claim 5, wherein the antibody is bound to a markerselected from the group consisting of fluorophores, biotin,radioisotopes, metals and enzymes.
 7. A kit for qualitative and/orquantitative detection of Flu virus comprising: a monoclonal antibody oran antigen-binding portion thereof that binds to PIV chimeric protein Laccording to claim 1, which acts as a capture or detection antibody,wherein detection antibody is conjugated to a marker for its detection;a solid support to which the antibody is attached; and reagents fordetecting the marker included in the detection antibody, such asfluorophores, biotin, radioisotopes, metals, and enzymes.
 8. The kit forqualitative and/or quantitative detection of Flu virus according toclaim 7, wherein the solid support is a membrane formed by one of thecompounds selected from the group consisting of nitrocellulose,cellulose, polyethylene and nylon.
 9. The kit for qualitative and/orquantitative detection of Flu virus according to claim 7, wherein thedetection of PIV is carried out with an immunochromatographic test,luminex, flow cytometry, immunofluorescence, radioimmunoanalysis,Western blot, Dot plot, ELISA, immunodiffusion or immunoprecipitation.10. (canceled)